HIV-1 coreceptor switching is driven by the high mutation rate of the envelope gene of the virus, with the result being expansion of the viral entry requirements from CD4 and CCR5 expression on the target cell to CD4 and CCR5 and/or CXCR4. This change in entry requirements precludes the use of CCR5 entry inhibitors as antiviral agents. Our proposed research program will examine changes in envelope sequence and function that precede coreceptor switching in subtype B HIV-1 infection, where expansion to CXCR4 use is common, and in subtype C infection, where it is uncommon. We will test a number of hypotheses based on published data from our laboratory. We will determine if loss of entry efficiency via CCR5 precedes mutations that confer CXCR4 use, and if mutations in regions other than V3 need to precede CXCR4-conferring mutations in V3 to maintain viral fitness. We will confirm that many more mutations in many more regions of envelope are necessary to confer CXCR4 use to subtype C HIV-1 isolates, and that these mutations change the entry preference for alternative coreceptors from FPRL1/FPR2 to CCR3. We will generate chimeric envelopes to demonstrate that mutations in V1/V2, C2, and C3 regions of envelope must occur prior to V3 changes to allow evolution of CXCR4 use. The extensive data on genotype and entry phenotype generated in these studies will be used to develop better genotypic predictors for coreceptor switching, potentially eliminating the need for slow and expensive phenotypic entry assays. We will explore the expression of the alternative coreceptors CCR3 and FPRL1/FPR2 and their use as intermediates in the coreceptor switching transition stage, and as potential alternatives for HIV-1 envelopes with mutations that confer resistance to CCR5 entry inhibitors. The goal of these studies is to be able to predict when coreceptor switching is likely to occur and to provide better guidance of the use of CCR5 inhibitors in a variety of clinical settings.